Interferon consensus sequence binding protein-deficient mice display impaired resistance to intracellular infection due to a primary defect in interleukin 12 p40 induction

Abstract

Mice lacking the transcription factor interferon consensus sequence binding protein (ICSBP), a member of the interferon regulatory factor family of transcription proteins, were infected with the intracellular protozoan, Toxoplasma gondii. ICSBP-deficient mice exhibited unchecked parasite replication in vivo and rapidly succumbed within 14 d after inoculation with an avirulent Toxoplasma strain. In contrast, few intracellular parasites were observed in wild-type littermates and these animals survived for at least 60 d after infection. Analysis of cytokine synthesis in vitro and in vivo revealed a major deficiency in the expression of both interferon (IFN)-gamma and interleukin (IL)-12 p40 in the T. gondii exposed ICSBP-/- animals. In related experiments, macrophages from uninfected ICSBP-/- mice were shown to display a selective impairment in the mRNA expression of IL-12 p40 but not IL-1alpha, IL-1beta, IL-1Ra, IL-6, IL-10, or TNF-alpha in response to live parasites, parasite antigen, lipopolysaccharide, or Staphylococcus aureus. This selective defect in IL-12 p40 production was observed regardless of whether the macrophages had been primed with IFN-gamma. We hypothesize that the impaired synthesis of IL-12 p40 in ICSBP-/- animals is the primary lesion responsible for the loss in resistance to T. gondii because IFN-gamma-induced parasite killing was unimpaired in vitro and, more importantly, administration of exogenous IL-12 in vivo significantly prolonged survival of the infected mice. Together these findings implicate ICSBP as a major transcription factor which directly or indirectly regulates IL-12 p40 gene activation and, as a consequence, IFN-gamma-dependent host resistance.

Figure 1

Progression of T. gondii infection in ICSBP^−/− versus control animals. Mice were infected by the i.p. route of infection with 20 ME49 cysts and cumulative mortality monitored. The data shown in (A and B) are pooled from three independent experiments and involve a total of 14 ICSBP^−/−, 15 ICSBP^+/+, 14 IL-12 p40 ko and 19 IFN-γ ko animals. (C) Qualitative analysis of the PEC present in wt (dark grey bars), ICSBP^−/− (black bars), IFN-γ ko (pale grey bars) and IL-12 p40 ko (hatched bars) animals at 5 d after infection with ME49. Cytospin smears of PEC were prepared and stained with Diff-Quik reagent, as described in Materials and Methods. The percentage of infected cells, large mononuclear cells (LMc; macrophages, monocytes, and blasting lymphocytes), small mononuclear cells (SMc; resting lymphocytes), polymorphonuclear cells (PMN) and eosinophils (Eos) were calculated. Uninfected mice of each of the four strains did not differ significantly in the composition of their PECs. Values shown are the mean ± SE of 6–12 infected animals per group. Statistically significant differences between the values observed in ICSBP^−/− and wt (*) or ICSBP^−/− and both IFN-γ ko^−/− and IL-12 p40 ko (**) samples are indicated (P ⩽0.05).

Figure 2

Kinetics of cytokine synthesis in infected wt (open squares) and ko (solid circle) animals. Serum, PECs, and spleen tissue were harvested from ICSBP^−/− and ICSBP^+/+ mice on days 0, 3, 5, and 7 after i.p. infection with ME49. IFN-γ (A) and IL-12 p40 (B) levels were measured by ELISA in diluted sera or in 72-h culture supernatants of single-cell suspensions as described in Materials and Methods. Data points are the mean ± SE for four individual mice. An asterisk indicates a statistically significant difference (P <0.05 by Student's t test) between the values observed in ICSBP^−/− and ICSBP^+/+ samples. The experiment shown is representative of three performed.

Figure 3

Con A–induced IFN-γ production by spleen cells from uninfected ICSBP^+/+, ICSBP^−/−, and IL-12 p40^−/− mice. Spleen cells were cultured with media alone (white bars) or Con A (black bars) at 5 μg/ml and the supernatants assayed for IFN-γ 72 h later. Cytokine synthesis (mean ± SE) is shown for four individual animals per group.

Figure 4

Kinetics of cytokine mRNA expression in splenic tissue from T. gondii-infected ICSBP^+/+ and ICSBP^−/− mice. (A) Splenic tissue was harvested from 4 individual mice at days 0, 3, 5, and 7 after parasite exposure from the same animals studied in Fig. 2. Extracted RNAs were subjected to quantitative RT-PCR analysis using primers specific for IL-12 p40, IL-12 p35, IFN-γ, IL-10, IL-4, and HPRT genes. (B) RNA from four individual mice were pooled and levels of TNFα expression were determined by RT-PCR as performed in (A). Similar results were attained in a second experiment.

Figure 4

Kinetics of cytokine mRNA expression in splenic tissue from T. gondii-infected ICSBP^+/+ and ICSBP^−/− mice. (A) Splenic tissue was harvested from 4 individual mice at days 0, 3, 5, and 7 after parasite exposure from the same animals studied in Fig. 2. Extracted RNAs were subjected to quantitative RT-PCR analysis using primers specific for IL-12 p40, IL-12 p35, IFN-γ, IL-10, IL-4, and HPRT genes. (B) RNA from four individual mice were pooled and levels of TNFα expression were determined by RT-PCR as performed in (A). Similar results were attained in a second experiment.

Figure 5

Defective IL-12 p40 protein and mRNA response in thioglycollate-elicited macrophage-enriched populations from ICSBP^−/− and ICSBP^+/+ mice. (A) IL-12 p40 protein synthesis in cultures of −/− (black bars) and +/+ (white bars) cells. (B) IL-12 p40 mRNA induction in peritoneal macrophages stimulated in vitro. PECs were collected from five mice that had been injected with thioglycollate 5 d earlier. Pooled adherent cells (macrophage enriched) were cultured in triplicate in the presence of SAC (1:1,000), LPS (200 ng/ml), RH (0.2 tachyzoites/cell), STAg (5 μg/ml), or media alone. Where indicated, cells were incubated with IFN-γ (200 U/ml) and/or anti–IL-4/10 mAbs (10 μg/ml each) for 2 h before the addition of stimuli and throughout the culture period. Cells were collected at 6 h for RNase protection assay and supernatants at 48 h for protein measurement by ELISA. Three μg of RNA were subjected to RNase protection assay to detect transcripts for IL-12 p40, IL-1α, IL-1β, IL-1Ra, and GAPDH. The experiment shown is representative of three performed.

Figure 5

Defective IL-12 p40 protein and mRNA response in thioglycollate-elicited macrophage-enriched populations from ICSBP^−/− and ICSBP^+/+ mice. (A) IL-12 p40 protein synthesis in cultures of −/− (black bars) and +/+ (white bars) cells. (B) IL-12 p40 mRNA induction in peritoneal macrophages stimulated in vitro. PECs were collected from five mice that had been injected with thioglycollate 5 d earlier. Pooled adherent cells (macrophage enriched) were cultured in triplicate in the presence of SAC (1:1,000), LPS (200 ng/ml), RH (0.2 tachyzoites/cell), STAg (5 μg/ml), or media alone. Where indicated, cells were incubated with IFN-γ (200 U/ml) and/or anti–IL-4/10 mAbs (10 μg/ml each) for 2 h before the addition of stimuli and throughout the culture period. Cells were collected at 6 h for RNase protection assay and supernatants at 48 h for protein measurement by ELISA. Three μg of RNA were subjected to RNase protection assay to detect transcripts for IL-12 p40, IL-1α, IL-1β, IL-1Ra, and GAPDH. The experiment shown is representative of three performed.

Figure 6

Survey of cytokine expression (TNF-α, IL-12 p40, IL-10, and IL-6) by RT-PCR analysis in thioglycollate elicited macrophage populations after stimulation with IFN-γ and/or SAC, STAg or LPS. (A) Two to four million peritoneal macrophages were cultured with (+) or without (−) pretreatment with IFN-γ at 200 U/ml for 2 h followed by the addition of STAg (5 μg/ml), SAC (1:1,000) or LPS (200 ng/ml) for 6 h. RNA was extracted as described in Materials and Methods and samples were subjected to quantitative RT-PCR as in Fig. 4. In lane 7, anti–IL-4 and anti–IL-10 mAbs (10 μg/ml each) were included in the cultures. ND indicates that the cDNA sample could not be evaluated. (B) Relative levels of mRNA induction were calculated by first normalizing the densitometry units for each cytokine to the densitometry units of the housekeeping gene (HPRT) for each sample. Second, a ratio of the values determined for ICSBP^−/− and ICSBP^+/+ samples was calculated. Values of 100% indicate equal expression between the two groups, those <100% signify ICSBP^−/− <ICSBP^+/+ and those >100% that ICSBP^−/− >ICSBP^+/+. (−) indicate that a value could not be determined because below the level of detection (see Media −IFN) or because the HPRT value for at least 1 sample was not available (see STAg–IFNγ). The experiment shown is representative of three performed.

Figure 6

Survey of cytokine expression (TNF-α, IL-12 p40, IL-10, and IL-6) by RT-PCR analysis in thioglycollate elicited macrophage populations after stimulation with IFN-γ and/or SAC, STAg or LPS. (A) Two to four million peritoneal macrophages were cultured with (+) or without (−) pretreatment with IFN-γ at 200 U/ml for 2 h followed by the addition of STAg (5 μg/ml), SAC (1:1,000) or LPS (200 ng/ml) for 6 h. RNA was extracted as described in Materials and Methods and samples were subjected to quantitative RT-PCR as in Fig. 4. In lane 7, anti–IL-4 and anti–IL-10 mAbs (10 μg/ml each) were included in the cultures. ND indicates that the cDNA sample could not be evaluated. (B) Relative levels of mRNA induction were calculated by first normalizing the densitometry units for each cytokine to the densitometry units of the housekeeping gene (HPRT) for each sample. Second, a ratio of the values determined for ICSBP^−/− and ICSBP^+/+ samples was calculated. Values of 100% indicate equal expression between the two groups, those <100% signify ICSBP^−/− <ICSBP^+/+ and those >100% that ICSBP^−/− >ICSBP^+/+. (−) indicate that a value could not be determined because below the level of detection (see Media −IFN) or because the HPRT value for at least 1 sample was not available (see STAg–IFNγ). The experiment shown is representative of three performed.

Figure 7

ICSBP^−/− mice can be stimulated exogenously to control T. gondii infection in vitro (A) and in vivo (B). (A) PECs were collected from naive mice (resident) or from animals injected i.p. with thioglycollate 5 d prior (thioglycollate elicited). Cells were pretreated with murine IFN-γ for 2 h. Cultures were infected with 0.2 RH tachyzoites per cell, pulsed 24 h later with [³H]uracil, and cultured overnight. Incorporated radioactivity was determined and expressed as mean CPM for triplicate cultures. Percentage killing is indicated above the bar for IFN-γ–treated cultures and was calculated using the formula indicated in Materials and Methods. Comparable results were observed in two additional experiments. (B) Survival was compared in ICSBP^−/− mice that were treated with PBS (10 mice per group) or rIL-12 (0.5 μg, 13 mice per group) over the first 5 d of infection. The results shown are pooled from two experiments.